Regulation of mycothiol metabolism by sigma(R) and the thiol redox sensor anti-sigma factor RsrA

Mycothiol (MSH) is the major thiol in Actinobacteria and plays a role analogous to that of glutathione. The biosynthetic pathway has been established in mycobacteria and is initiated by the glycosyltransferase MshA. A key mycothiol-dependent detoxification pathway utilizes the amidase (Mca) to cleave mycothiol S-conjugates to produce GlcN-Ins and a mercapturic acid excreted from the cell. How expression of mycothiol genes is regulated in mycobacteria has been unclear so the report in this issue by Park and Roe showing that in Streptomyces coelicolor the redox controlled anti-sigma factor RsrA that binds the regulator sigma(R) controls key elements of mycothiol metabolism is a major advance. Conditions that deplete thiols are shown to induce directly expression of sigR, rsrA, mshA and mca, as well as the thioredoxin reductase-thioredoxin system, generating an autoregulatory cycle that persists until the thiol-depleting condition is alleviated. Evidence for indirect induction of mshB-D to support mycothiol biosynthesis is also presented. It was shown in vitro that mycothiol, like reduced thioredoxin and dithiothreitol, can reduce oxidized RsrA to activate its binding to sigma(R). These studies establish for the first time how mycothiol metabolism is regulated to cope with stress from thiol reactive toxins.     

Determinants of redox sensitivity in RsrA, a zinc-containing anti-sigma factor for regulating thiol oxidative stress response

Various environmental oxidative stresses are sensed by redox-sensitive regulators through cysteine thiol oxidation or modification. A few zinc-containing anti-sigma (ZAS) factors in actinomycetes have been reported to respond sensitively to thiol oxidation, among which RsrA from Streptomyces coelicolor is best characterized. It forms disulfide bonds upon oxidation and releases bound SigR to activate thiol oxidative stress response genes. Even though numerous ZAS proteins exist in bacteria, features that confer redox sensitivity to a subset of these have been uncharacterized. In this study, we identified seven additional redox-sensitive ZAS factors from actinomycetes. Comparison with redox-insensitive ZAS revealed characteristic sequence patterns. Domain swapping demonstrated the significance of the region K(33)FEHH(37)FEEC(41)SPC(44)LEK(47) that encompass the conserved HX(3)CX(2)C (HCC) motif. Mutational effect of each residue on diamide responsive induction of SigR target genes in vivo demonstrated that several residues, especially those that flank two cysteines (E39, E40, L45, E46), contribute to redox sensitivity. These residues are well conserved among redox-sensitive ZAS factors, and hence are proposed as redox-determinants in sensitive ZAS. H37A, C41A, C44A and F38A mutations, in contrast, compromised SigR-binding activity significantly, apparently affecting structural integrity of RsrA. The residue pattern around HCC motif could therefore serve as an indicator to predict redox-sensitive ZAS factors from sequence information.     

The Role of zinc in the disulphide stress-regulated anti-sigma factor RsrA from Streptomyces coelicolor

The regulation of disulphide stress in actinomycetes such as Streptomyces coelicolor is known to involve the zinc-containing anti-sigma factor RsrA that binds and inactivates the redox-regulated sigma factor sigmaR. However, it is not known how RsrA senses disulphide stress nor what role the metal ion plays. Using in vitro assays, we show that while zinc is not required for sigmaR binding it is required for functional anti-sigma factor activity, and that it plays a critical role in modulating the reactivity of RsrA cysteine thiol groups towards oxidation. Apo-RsrA is easily oxidised and, while the Zn-bound form is relatively resistant, the metal ion is readily expelled when the protein is treated with strong oxidants such as diamide. We also show, using a combination of proteolysis and mass spectrometry, that the first critical disulphide to form in RsrA involves Cys11 and one of either Cys41 or Cys44, all previously implicated in metal binding. Circular dichroism spectroscopy was used to follow structural changes during oxidation of RsrA, which indicated that concomitant with formation of this critical disulphide bond is a major restructuring of the protein where its alpha-helical content increases. Our data demonstrate that RsrA can only bind sigmaR in the reduced state and that this state is stabilised by zinc. Redox stress induces disulphide bond formation amongst zinc-ligating residues, expelling the metal ion and stabilising a structure incapable of binding the sigma factor.     

Novel Mycobacterium tuberculosis anti-sigma factor antagonists control sigmaF activity by distinct mechanisms

The aetiological agent of tuberculosis, Mycobacterium tuberculosis, encodes 13 sigma factors, as well as several putative anti-, and anti-anti- sigma factors. Here we show that a sigma factor that has been previously shown to be involved in virulence and persistence processes, sigmaF, can be specifically inhibited by the anti-sigma factor UsfX. Importantly, the inhibitory activity of UsfX, in turn, can be negatively regulated by two novel anti-anti-sigma factors. The first anti-anti-sigma factor seems to be regulated by redox potential, and the second may be regulated by phosphorylation as it is rendered non-functional by the introduction of a mutation that is believed to mimic phosphorylation of the anti-anti-sigma factor. These results suggest that sigmaF activity might be post-translationally modulated by at least two distinct pathways in response to different possible physiological cues, the outcome being consistent with the bacteria's ability to adapt to diverse host environments during disease progression, latency and reactivation.     

The Bacillus subtilis sigmaW anti-sigma factor RsiW is degraded by intramembrane proteolysis through YluC

The Bacillus subtilis sigma(W) regulon is induced by different stresses such as alkaline shock, salt shock, phage infection and certain antibiotics that affect cell wall biosynthesis. The activity of the alternative, extracytoplasmic function (ECF) sigma factor sigma(W) is modulated by a specific anti-sigma factor (RsiW or YbbM) encoded by the rsiW (ybbM) gene located immediately downstream of sigW. The RsiW membrane topology was determined, and a specific reporter system for RsiW function was constructed. Experiments using the yeast two-hybrid system suggested a direct interaction of sigma(W) with the cytoplasmic part of RsiW. Analysis of truncated forms of the RsiW protein revealed that sigma(W) induction by alkaline shock is dependent on both the transmembrane and the extracytoplasmic domain of RsiW. Western blot and pulse-chase experiments demonstrated degradation of RsiW after an alkaline shock. A B. subtilis mutant strain deleted for the Escherichia coli yaeL orthologue yluC, encoding a transmembrane protease, was defective in inducing a sigma(W)-controlled promoter after alkaline shock and accumulated a membrane-bound truncated form of RsiW, suggesting that the activity of sigma(W) is controlled by the proteolysis of RsiW by at least two different proteolytic steps.     

Interactions between the Rhodobacter sphaeroides ECF sigma factor, sigma(E), and its anti-sigma factor, ChrR

Rhodobacter sphaeroides sigma(E) is a member of the extra cytoplasmic function sigma factor (ECF) family, whose members have been shown to regulate gene expression in response to a variety of signals. The functions of ECF family members are commonly regulated by a specific, reversible interaction with a cognate anti-sigma factor. In R.sphaeroides, sigma(E) activity is inhibited by ChrR, a member of a newly discovered family of zinc containing anti-sigma factors. We used gel filtration chromatography to gain insight into the mechanism by which ChrR inhibits sigma(E) activity. We found that formation of the sigma(E):ChrR complex inhibits the ability of sigma(E) to form a stable complex with core RNA polymerase. Since the sigma(E):ChrR complex inhibits the ability of the sigma factor to bind RNA polymerase, we sought to identify amino acid substitutions in sigma(E) that altered the sensitivity of this sigma factor to inhibition by ChrR. This analysis identified single amino acid changes in conserved region 2.1 of sigma(E) that either increased or decreased the sensitivity of sigma(E) for inhibition by ChrR. Many of the amino acid residues that alter the sensitivity of sigma(E) to ChrR are located within regions known to be important for interacting with core RNA polymerase in other members of the sigma(70) superfamily. Our results suggest a model where solvent-exposed residues with region 2.1 of sigma(E) interact with ChrR to sterically occlude this sigma factor from binding core RNA polymerase and to inhibit target gene expression.